Center for Biophysics and Computational Biology, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA; Department of Physics and Center for the Physics of Living Cells, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA; Howard Hughes Medical Institute, Urbana, IL 61801, USA. Electronic address: tjha@illinois.edu.

Abstract

Bacteriophage T7 gp4 serves as a model protein for replicative helicases that couples deoxythymidine triphosphate (dTTP) hydrolysis to directional movement and DNA strand separation. We employed single-molecule fluorescence resonance energy transfer methods to resolve steps during DNA unwinding by T7 helicase. We confirm that the unwinding rate of T7 helicase decreases with increasing base pair stability. For duplexes containing >35% guanine-cytosine (GC) base pairs, we observed stochastic pauses every 2-3 bp during unwinding. The dwells on each pause were distributed nonexponentially, consistent with two or three rounds of dTTP hydrolysis before each unwinding step. Moreover, we observed backward movements of the enzyme on GC-rich DNAs at low dTTP concentrations. Our data suggest a coupling ratio of 1:1 between base pairs unwound and dTTP hydrolysis, and they further support the concept that nucleic acid motors can have a hierarchy of different-sized steps or can accumulate elastic energy before transitioning to a subsequent phase.

The Unwinding Rate of T7 Helicase Depends on Base Pair Stability(A) T7 gp4 was loaded on a 40 bp DNA with (dT)n tails containing donor (Cy3) and acceptor (Cy5) dyes, and bound to a PEG-coated surface via biotin-neutravidin interaction.(B) Cy3 and Cy5 intensity traces during unwinding for one molecule on mixed, 100% AT, and 80% GC sequences (top panel); calculated FRET efficiency versus time for the fluorescence intensity traces (bottom panel).(C) Dwell-time histograms during unwinding. The arrows on the FRET traces indicate the intervals at which the dwell times were measured; 50 molecules were used to build the histograms. The data are representative of multiple experiments.(D) Unwinding time (red circles) and rate (blue triangles) versus base pair stability. Five sequences were used to plot the graph ().See also .

Calibration of the Number of Base Pairs Unwound to FRET(A) DNA construct schematics.(B) Representative calculated FRET efficiency versus time traces for 10 AT and 30 GC substrates (right and left panels).(C) FRET histogram. The arrows indicate the region of the FRET values used to build the histogram; >50 molecules were used to build the histogram. The histogram peaks at 0.3 FRET.

Analysis of Step Size and Stepping Kinetics(A) DNA sequences are made of repeats of n AT base pairs followed by n GC base pairs where n=3, 4, or 5.(B) A step-finding algorithm was used to measure FRET values and dwell times of the pauses for a single molecule. Three sequences with different GC content (from top to bottom: 48%, 50%, and 80%) were used for analysis. The step size was measured by counting the number of steps (n) until FRET reached a value of 0.3, and then dividing 10 by n.(C) Histogram of the number of steps that occurred for each substrate.(D) FRET histograms during unwinding; >50 molecules were used to build the histograms.(E) FRET values obtained from 80 molecules from each substrate were combined to make the transition density plot (TDP).(F) Gamma distribution fitting of the collected dwell times at each pause for >50 molecules.

Dwell Time of Pauses with Lower dTTP Concentration and Backward Movements(A) Percentage of unwound molecules as a function of [dTTP].(B) Unwinding time from high FRET to low FRET as a function of [dTTP].(C) A step-finding algorithm was used to measure FRET values and dwell times of each pause for a single molecule. Experiments were performed using 100 μM dTTP on an 80% GC substrate.(D) Gamma distribution fitting of the collected dwell times at each pause for >50 molecules.(E) Cy3 and Cy5 intensity trace during unwinding on an 80% GC sequence, depicting backward movements by the T7 helicase during unwinding at 100 μM [dTTP] (top left panel), and Cy3 and Cy5 intensity trace displaying unwinding without backward movements at 1 mM [dTTP] (bottom left panel). The lines indicate the areas selected to calculate cross-correlation. Top and bottom right panels: calculated FRET efficiency versus time for the fluorescence intensity trace. Smoothed curves that were used for cross-correlation analysis are depicted in blue (see ).(F) Percentage of backward-movement traces as a function of [dTTP].(G) Average cross-correlation curve from 50 molecules for 100 μM (left panel) and 1 mM dTTP (right panel).See also .

T7 Helicase Unwinding Model(A) dsDNA unwinding of AT base pairs. Only the DNA-binding loop of the rearmost position is shown, which is released upon dTDP release. Next, dTTP binds and the released loop binds the DNA at the foremost position. The fast melting of AT base pairs enables the enzyme to move forward.(B) dsDNA unwinding of GC base pairs. Slow melting of GC base pairs slows down the enzyme. When the enzyme encounters GC base pairs, reengagement of the disengaged loop with the DNA ahead is hindered due to steric mismatch between dsDNA and central helicase pore. Subsequent dTTP hydrolysis and product release by the next rearmost subunits build enough strain in the system to eventually cause 2–3 bp unwinding in a burst.(C) dsDNA unwinding at low dTTP concentrations. The DNA-binding loop does not readily bind to the DNA because the subunit remains in the dTTP unbound state. Subsequent dTTP hydrolysis and dTDP release at other subunits leads to the detachment of all of the DNA-binding loops and backward movements by the enzyme, and the DNA rezips. Once a new set of contacts is created by the enzyme on the tracking strand, the enzyme can move forward and unwind DNA.